Nucleotides within the liquid film lining the epithelia of the airways control the mucociliary (MCC) clearance process that removes inhaled noxious materials. ATP, UTP, UDP, and adenosine activate a subset of purinergic receptors that regulate ion secretion, increase ciliary beat frequency, and promote mucin secretion. In addition, UTP, ATP, and UDP-glucose are potent neutrophil chemoattractants, suggesting that nucleotides released onto airway surfaces may induce neutrophil migration in response to airway stress. Therefore, understanding the mechanism(s) of nucleotide release by airway epithelia has important pathophysiological and therapeutic implications for the lung. Circumstantial evidence exists for two modes of ATP release from epithelial cells onto the airway surface: (i) ion channels and/or transporters and (ii) vesicles. However, whether nucleotides are released from the cytosol, from vesicles, or both compartments is not unambiguously known. Recognition that airway epithelial cells release UDP-sugars, donor substrates of glycosylation reactions in the endoplasmic reticulum and Golgi lumen, in addition to ATP, suggests that nucleotides entering these organelles may be released by exocytosis. Preliminary data illustrating that enhanced mucin secretion by cultured goblet-like cells is accompanied by release of UDP-glucose and ATP suggest that nucleotide release by goblet cells reflects an exocytotic process associated with mucin secretion. Preliminary data also suggests that NMP antiporters mediate the release of cytosolic UDP-sugars in non-goblet cells. This application outlines a plan to define the role of vesicle and mucin granule exocytosis and of antiporter mechanisms in the release of nucleotides by airway epithelia. To accomplish this goal, we propose the following Specific Aims: (1) to test the hypothesis that NTP/NDP release is vesicular, (2) to test the hypothesis that nucleotide/NMP antiporters facilitate nucleotide release, and (3) to delineate the relationship between nucleotide release and mucin secretion. We will assess the extent to which nucleotide release reflects vesicle exocytosis, is modified by Golgi nucleotidases, involves Golgi and/or cell surface translocators, and is mechanistically associated with mucin secretion. Completion of these studies will establish mechanism(s) for the physiologically important process of nucleotide release, and accordingly will delineate potential drug targets for airway diseases associated with poor MCC clearance.